Bacterial Peroxidase on Electrochemically Reduced Graphene Oxide for Highly Sensitive H2O2 Detection

Abstract Peroxidase enzymes enable the construction of electrochemical sensors for highly sensitive and selective quantitative detection of various molecules, pathogens and diseases. Herein, we describe the immobilization of a peroxidase from Bacillus s. (BsDyP) on electrochemically reduced graphene oxide (ERGO) deposited on indium tin oxide (ITO) and polyethylene terephthalate (PET) layers. XRD, SEM, AFM, FT‐IR and Raman characterization of the sensor confirmed its structural integrity and a higher enzyme surface occupancy. The BsDyP‐ERGO/ITO/PET electrode performed better than other horseradish peroxidase‐based electrodes, as evinced by an improved electrochemical response in the nanomolar range (linearity 0.05–280 μM of H2O2, LOD 32 nM). The bioelectrode was mechanically robust, active in the 3.5–6 pH range and exhibited no loss of activity upon storage for 8 weeks at 4 °C.


Expression and purification of the peroxidase from Bacillus subtilis (BsDyP) and activity test
The synthetic gene encoding the BsDyP peroxidase bearing an additional poly-histidine tag was designed according to literature, [1] and cloned in E. coli BL21 DE3 cells. Enzyme expression and purification were performed as follows. For recombinant expression, 800 mL of LB medium supplemented with ampicillin (100 µg/mL) were inoculated with 15 mL of an E. coli overnight culture harboring the desired plasmid DNA. Cells were grown at 37 °C until an OD600 of 0.8-0.9 was reached and expression of protein was induced by the addition of IPTG (0.5 mM), 5-aminolevulinic acid (0.5 mM) and ferrous sulfate monohydrate (100 µM). Protein expression was carried out overnight at 20 °C. After harvesting of the cells (4 °C, 8 x 103 rpm, 10 min), the remaining cell pellet was re-suspended in lysis buffer (50 mM KH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) prior to cell disruption by ultrasonication. Protein purification was performed by Ni-NTA affinity chromatography using prepacked Ni-NTA HisTrap HP columns previously equilibrated with lysis buffer. After loading of the filtered lysate, the column was washed with sufficient amounts of washing buffer (50 mM KH2PO4, 300 mM NaCl, 25 mM imidazole, pH 8.0); next, bound protein was recovered with elution buffer (50 mM KH2PO4, 300 mM NaCl, 200 mM imidazole, pH 8.0). After SDS-PAGE, fractions containing the desired proteins in a sufficient purity (> 90%) were pooled and dialyzed overnight against K2HPO4/KH2PO4 buffer (50 mM, pH 7.5) and then concentrated using Centripreps (Millipore). Purified BsDyP enzyme was aliquoted, shock-frozen in liquid nitrogen and stored at -80 °C. The final concentration of the protein was determined at 280 nm (ε280 = 42525 M -1 cm -1 ). A typical protein yield of 41 mg L -1 of cell culture was obtained with an Rz value (Asoret,408/A280) of 0.54 (approximately 38% of the protein was saturated with hemin; protein concentration: 427 µM, 20 mg mL -1 total protein).

Preparation of stock solutions
Solutions of BsDyP (427 µM) was freshly diluted in PBS buffer (50 mM, pH 7.0) and henceforth named as solution A. Stock solution of H2O2 was prepared in water and stored at 4 °C. This stock solution was further diluted to prepare different concentrations of the analyte (H2O2). Solutions of EDC (0.4 M) and NHS were prepared freshly in deionized water.

Procedure for the synthesis of graphene oxide (GO) dispersion in water
Graphene oxide (GO) was prepared by modified Hummers' process. [2] Graphite powder was taken in a conical flask. NaNO3 and H2SO4 were added in the flask already containing the graphite powder and stirred at 0 °C for 30 minutes (henceforth this solution is named as solution B). Next, KMnO₄ was added slowly to the solution B while stirring at room temperature. Solution was left for over-night stirring (henceforth, this solution is named as solution C). The color of the solution was green. Next day, H2O was added dropwise to the solution C while stirring and the mixture was left for 30 minutes to maintain the temperature (henceforth, this solution is named as solution D). The color of solution changes from green to brown.
Later, when the temperature of solution reached room temperature, H2O2 (30% v v -1 ) was added to the solution D while stirring and the color changed from brown to yellow. Thus, GO was prepared as shown in Figure S2. The prepared GO solution was washed with HCl (5% v v -1 ) using centrifuge machine to remove the impurities. After washing with HCl, the solution was further washed with distilled water several times to change the pH from acidic to neutral. Finally, prepared GO water solution (1 mg mL -1 ) was sonicated for 2 hours.

Fabrication of electrochemically reduced graphene oxide (ERGO) film on ITO electrode
The ITO/PET sheet was cut into electrodes of 1 cm x 2 cm dimensions. Electrodes were cleaned with acetone, ethanol, isopropyl alcohol and water. The ITO/PET electrodes were dried by N2 blower. The above prepared GO suspension (1 mg mL -1 ) was reduced on pre-cleaned ITO surface with application of a constant potential (-1.5 V) at neutral pH via chronoamperometric technique using a three-electrode system with ITO/PET as a working electrode (W), platinum (C) wire as an auxiliary electrode and Ag/AgCl as a reference electrode (R) as shown in Figure S3. Figure S3 shows the diagrammatic representation of three electrode system and camera captured image of the developed electrode.

Immobilization of BsDyP onto ERGO/ITO electrode
BsDyP peroxidase was covalently attached to ERGO/ITO electrode via carbodiimide chemistry. For immobilization of the enzyme, 30 μL of solution A was mixed with EDC (0.4 M) and NHS (0.1 M). This enzyme mixed solution was added dropwise onto 0.25 cm 2 of ERGO/ITO electrode and kept in a humid chamber for 4 h. Next, the fabricated bioelectrode (BsDyP-ERGO/ITO) was washed with PBS solution (50 mM, 0.9% NaCl, pH 7.0) containing 0.05% Tween 20 to remove any unbound enzymes. The prepared BsDyP-ERGO/ITO bio-electrodes were refrigerated at 4 °C when not in use.

Optimization for BsDyP concentration in fabricating BsDyP-ERGO/ITO bioelectrode
The BsDyP-ERGO/ITO was optimized by pursuing measurements with four different concentrations of BsDyP stock solution in PBS buffer (i.e., 0.5 mg mL -1 , 1.0 mg mL -1 , 1.5 mg mL -1 , 2.0 mg mL -1 ). The amount and concentration of BsDyP was optimized by drop-casting the different BsDyP solutions containing 0.4 M EDC and 0.1 M NHS over 0.25 cm 2 area of ERGO/ITO electrodes (named as electrodes B1, B2, B3 and B4 for 0.5 mg mL -1 solution, 1.0 mg mL -1 solution, 1.5 mg mL -1 solution and 2.0 mg mL -1 solution, respectively). After drop-casting the electrodes were placed in a humid chamber for 4 hours. The CV scans were recorded in acetate buffer (pH 4, 0.2 M) containing 5 mM [Fe(CN)6] 3−/4− at scan rate of 50 mVs -1 . The determined change in anodic peak currents (Ipa) of bio-electrodes B1, B2, B3 and B4 were plotted in a bar diagram (Fig. S4a). The maximum magnitude of peak current was observed for B2 (i.e., 1.0 mg mL -1 ). Next, the B1, B2, B3 and B4 electrodes were tested for the detection of H2O2 (named as H1, H2, H3 and H4, respectively). CV scans were recorded at scan rate of 50 mVs  Figure S3b). The observation of maximum change in electronic signals was observed for case H2. The large change in Ipa recorded for this BsDyP-H2O2 interaction suggested enhanced capability for detection of H2O2. Therefore, the optimum performance of the BsDyP-ERGO/ITO bioelectrode is obtained by using a 1 mg mL -1 concentration of the enzyme. On the one hand, for lower enzyme concentration, there is not enough enzymes to catalyze the optimal H2O2 reduction. On the other hand, when concentration is increased, the decrease in the anodic peak current is due to hindrance in charge transfer at the anode.  [3] and the BsDyP from this study (without poly-histidine purification tag) Fig. S5. Sequence alignment between DyP from Bacillus subtilis (PDB 6KMN) [3] and the BsDyP from this study. The initial amino acid chain is missing in PDB 6KMN comparing with the BsDyP from this study. PDB 6KMN and the BsDyP from this study also differ for further 12 amino acid residues.

Generation of homology model of BsDyP
The generation of the homology model was carried out using the YASARA [4] (Version 19.12.14.W.64) homology model building protocol, [5] which involves multi-template structural model generation. Since the linear amino acid sequence of the target protein was the only given input, one template was manually provided namely the X-ray crystal structure of another DyP from B. subtilis that was recently crystallized and biochemically characterized (PDB 6KMN). [3] The other possible templates were identified by running 3 PSI-BLAST [6] iterations to extract a position specific scoring matrix (PSSM) from UniRef90, [7] and then searching the PDB for a match with an E-value below the entered homology modelling cut-off of 0.5. A maximum of five templates was allowed, therefore the one manually provided plus additional four selected through the bioinformatics search. To aid alignment correction and loop modelling, a secondary structure prediction for the target sequence had to be obtained. This was achieved by running PSI-BLAST to create a target sequence profile and feeding it to the PSI-Pred [8] secondary structure prediction algorithm. For each of the found templates, models were built. Either a single model per template was generated, when the alignment was certain, or a number of alternative models were generated, when the alignment was ambiguous. A maximum of 50 conformations per loop were explored. A maximum of 10 residues were added to the termini. Finally, YASARA tried to combine the best parts of the generated models to obtain a hybrid model, with the intention of increasing the accuracy beyond each of the contributors. The quality of the models was evaluated by use of Z-score. [9] A Z-score describes how many standard deviations the model quality is away from the average high-resolution X-ray structure. The overall Zscore for the model was calculated as the weighted averages of the individual Z-scores using the formula: The overall score thus captures the correctness of backbone (Ramachandran plot) and side-chain dihedrals as well as packing interactions. Notably, the obtained model was already of high quality; therefore, molecular dynamic refinement simulation was not necessary.
Amino acid sequence of BsDyP:

General information
The BsDyP peroxidase immobilized on ERGO-deposited ITO surfaces were characterized for purity and optimized for observing uniform layers of ERGO flakes using various advanced nanotechnology tools including Fourier transform infrared (FTIR) spectroscopy (Gladi ATR, Pike technologies). For the FTIR of GO/ITO, sample was prepared by drop-casting GO solution (1 mg mL -1 ) onto the ITO glass. We used ITO/glass instead of PET to avoid the noise due to PET peaks in the ERGO/ITO sample. Raman, XRD, CV (Auto lab potentiostat/galvanostat), AFM studies were also performed. For Raman and XRD characterization, samples were also prepared on ITO/glass for the same reason as above. Scanning electron microscopy (SEM) images were acquired with a Zeiss Auriga scanning electron microscope. Before imaging, the samples were sputter-coated with platinum/iridium.

XRD studies
The structural differences between graphite, GO and ERGO/ITO can clearly be seen by comparing their X-Ray Diffraction (XRD) spectra (Supporting information, Fig. S6). In raw graphite, the crystalline peak is found at 2θ = 26.46° (lattice spacing of 0.34 nm) that is its characteristic (002) diffraction peak. After the oxidation to GO, the peak shifts to a lower angle at 2θ = 10.7° (lattice spacing of 0.81 nm). This increase in interlayer spacing is ascribed to the intercalation of water and the presence of oxygen functionalities (epoxide, carboxyl, hydroxyl) on the carbon basal plan sheet. In ERGO/ITO the diffraction peak at 10.7° disappeared and a new peak appeared at 2θ = 26.7° (lattice spacing of 0.34 nm) as a result of the successful electrochemical reduction.     where Ipa is the current intensity in Ampere and Ct is the concentration of H2O2 (μM).

Electrochemical characterization
The R 2 coefficient of the linear regression was 0.985.